Our aim is to understand the function, detailed structure and immunologic properties of nuclear macromolecules which are the targets of autoimmune antibodies in the rheumatic diseases. This proposal focuses on two antigens: a class of nuclear RNA-protein (nRNP) complexes and a basic chromosomal protein of molecular weight 70,000 (Scl-70). Their corresponding antibodies occur in mixed connective tissue disease (MCTD) and scleroderma, respectively. We previously isolated the nRNP antigen from mammalian nuclei and defined its molecular composition (1). The core of the antigen is composed of a protein of molecular weight 13,000 (P13) and RNA. The function of the RNA remains unknown. One of the objectives of this proposal is to determine if the RNA of the antigen is functionally associated with other classes of nuclear RNA. We have developed a protocol involving sucrose density sedimentation and immunoaffinity chromatography which yields core antigen free of contaminating ribonucleoprotein. The RNA of the core antigen will be analyzed by gel electrophoresis and T1 and RNase A fingerprinting. This RNA will be compared to other nuclear RNA classes by nucleic acid hybridization to determine the extent of homology or complementarity. As an outgrowth of the nRNP project, we biochemically isolated and identified a scleroderma-associated antigen and named it Scl-70. The primary and higher order structure of Scl-70 will be investigated by peptide mapping, amino acid analysis and renaturation studies. These experiments will be coordinated with efforts to identify its important antigenic features. Scl-70 is associated with a protein kinase activity and is itself phosphorylated. The effects of phosphorylation on its antigenic activity will be investigated. Another objective is to determine if the kinase activity is a property of Scl-70 or of a minor adhering protein. A third objective is to identify the preferred substrates of the enzyme. We also plan to investigate the immunoglobulin subclass-composition of anti-Scl-70 antibodies. The two macromolecular systems under investigation are viewed as model systems for understanding the immune phenomenon associated with rheumatic disease.